Nutrient Remineralization by Ramicrusta

Background

Brief Introduction:

The rapid proliferation of the red encrusting macroalgal Ramicrusta in the Caribbean is raising concerns about its ecological impacts, particularly its ability to outcompete foundational benthic species, inhibit invertebrate recruitment, and overgrow living coral colonies, often leading to coral mortality (Eckrich & Engel, 2013; Edmunds et al., 2019). Coral genera exhibit varying levels of susceptibility to Ramicrusta colonization, with Orbicella sp. among the most vulnerable and experiencing high alga overgrowth rates (Hollister et al., 2021; Fig. 1).

There’s been considerable evidence illustrating Ramicrusta’s tendency to interact with and overgrow live corals (Eckrich & Engel, 2013; Ballantine et al., 2016; Edmunds et al., 2019; Hollister et al., 2021), yet there remains a disconnect when it comes to understanding the factors contributing to this relationship. As macroalgal growth is generally restricted by nutrient availability, particulary in these oligotrophic water, I hypothesize that Ramicrusta may be deriving nutrients from the coral itself, fueling these interactions and promoting its own growth.

$Description$
Fig. 1: R. textilis overgrowing an O. annularis colony (photo: Abigail Gretta).

Application of Stable Isotope Analysis:

Based on predictable isotope fractionation patterns (Fry, 1988), the ability of the alga to remineralize coral nutrients can be evaluated through bulk stable isotope analyses. Fractionation refers to the preferential use of the lighter isotope (¹²C, ¹⁴N) during metabolic processes, concentrating the heavier form in the organism’s tissues (¹³C, ¹⁵N) (Rounick & Winterbourn, 1986; Fry, 1988; Cohen & Fong, 2004). In heterotrophic consumers, the ratio of heavy to light isotopes for carbon (C) and nitrogen (N) generally enrich by 1‰ and 3‰ per trophic level, respectively (Fry, 1988). These ratios, or isotope signatures, are represented by the delta notation in parts per thousand ($\delta^{13}$C, $\delta^{15}$N). This unique aspect of isotope fractionation provides insight into a consumer’s food source ($\delta^{13}$C) and trophic position ($\delta^{15}$N) (Rounick & Winterbourn, 1986; Post et al., 2002). However, primary producers typically exhibit a lack of fractionation (Gartner et al., 2002; Cohen & Fong, 2004; Strait et al., 2021). Thus, both the C and N signatures generally reflect their source. As a result, macroalgal bioassays serve as powerful proxies for nutrient studies in oligotrophic environments because tissue analyses can trace nutrient sourcing ($\delta^{13}$C, $\delta^{15}$N), availability (C%, N%), and limitations (C:N) in the surrounding water (Amato et al., 2016; Amato et al., 2018; Strait et al., 2021).

In theory, if Ramicrusta species are remineralizing coral tissues, the C and N isotope signatures and percentage of the algae should be higher due to the trophic enrichment (Fry, 1988; Muscatine et al., 2005). Additionally, the C and N percentages may be enhanced in the alga as a result of additional nutrients derived from the coral tissue, which would, in turn, reduce nutrient limitations and lower the C:N ratio. (Amato et al., 2016). However, baseline tissue nutrient analyses on the genus Ramicrusta are lacking; therefore, we will need to include two upright geniculate calcifying macroalgae species into our sampling to offer context into Ramicrusta’s nutrient parameters. The rhizophytic green alga Halimeda opuntia and epilithic red alga Jania adhereans will be prioritized because they are prominent calcifiers within the region and their nutrient contents have been established for oligotrophic reef systems (Fong et al., 2003; Koch et al., 2023).

The following are the preliminary methods, results, and conclusions from a pilot study conducted on September 3, 2024.

Methods

Site Description:

Sample collection occurred on the leeward side of Flat Cay, an offshore 9 m depth reef along the southwest side of St. Thomas, U.S. Virgin Islands (Fig. 2). This site was selected because of its high abundance of R. textilis (46.0% ± 5.9; Hollister et al., 2021) and high frequency of interactions between the alga and Orbicella annularis (personal observation). To characterize the abiotic conditions during algal and coral sampling, a temperature logger (Hobo Pendant) and PAR meter were deployed a month prior to the collection time and retrieved on the final day of sampling.

Alga Collection:

Open-circuit SCUBA was used for all algal collections. Replicates of each algal sample was randomly collected by hand, with a minimum of five meters between samples. The apical regions of H. opuntia and J. adhaerens (~ 5 cm) were cleanly removed from its thallus (n = 10 samples per species). R. textilis was collected from ten rocks (control; n = 10) and ten O. annualaris colonies that appeared visually healthy. For each substrate, R. textilis was brushed of epiphytes and chiseled at two locations from the same contiguous alga thallus (n = 2 R. textilis samples per substrate). One sample was collected from the margin of algal growth and the other was at a linear distance away from the margin, with distances between samples measured (~ 2 to 3 cm) and photographed (Fig. 3). This sampling strategy will distinguish the alga’s nutrient parameters during direct interaction with living coral to those in non-interacting regions, with the algal samples from bare substrate serving as controls for both conditions. Algal replicates will be placed in individual plastic bags at depth with ambient seawater. Samples will be transported to the laboratory in a dark container to minimize physiological stress and processed immediately.

Isotope Preparation:

Macroalgae were rinsed with deionized water (DI) to remove any external contaminants (i.e., invertebrates and epiphytes) and thoroughly dried with paper towels (Strait et al., 2021). Samples were loosely parceled in combusted aluminum foil with each sample code and dried at 60℃ to constant weight in a drying oven (24-36 hours). With a clean mortar and pestle (ethanol and DI rinse between samples), algae were ground to a fine powder and carefully packaged in 1.5 mL Eppendorf tubes (Strait et al., 2021). Stable isotope analyses were processed by an Elemental Analyzer Delta V at the University of Hawai’i at Mānoa. Dual stable isotope analyses were performed because acidification has the potential to degrade $\delta^{15}$N and unaltering calcified tissue can skew $\delta^{13}$C (Strait & Spalding, 2021).

Data Analysis:

All data were tested for homogeneity and normality. Log transformations were used when data violated ANOVA assumptions. Tissue nutrients ($\delta^{15}$N, % N, and C:N) were assessed using two-factor ANOVA (factors: Substrate, Growth Region, and the interaction of the two). Tukey’s honestly significant difference post hoc test was applied to show pairwise comparisons, with distinct letters on graphs denoting significant differences.

Results

Effects of Remineralization:

A two-factor ANOVA with a log transformation revealed significant differences in the N signatures of Ramicrusta for substrate type (p-value = 0.04978) and growth region (p-value = 0.01112), with no significant interaction effect between the two factors. Marginal (mean $\delta^{15}$N ± SEM = 1.66 ‰ ± 0.10) and non-marginal (mean $\delta^{15}$N ± SEM = 1.81 ‰ ± 0.06) samples of R. textilis growing on O. annularis had higher N signatures compared to their corresponding controls (mean $\delta^{15}$N ± SEM = 1.36 ‰ ± 0.13 and 1.78 ‰ ± 0.13, respectively; Fig. 5). Nitrogen signatures were consistently lower in marginal samples than in non-marginal samples across both substrates.

Fig. 5: Boxplot of the N signatures from R. textilis growing on O. annularis (n = 10 colonies) and bare substrate (i.e., control; n = 10 rocks), with a marginal and non-marginal samples collected from each substrate type. A two-factor ANOVA found significant differences between substrate (p-value = 0.04978) and growth region (p = 0.01112), but not the interaction effect of the two factors. Letters denote significant differences from a Tukey’s post hoc test.

In contrast, the N content of R. textilis exhibited the opposite pattern observed in the alga’s $\delta^{15}$N values for substrate type and growth region. The marginal algal samples collected from O. annularis colonies had significantly higher N percentages than the non-marginal replicates (two-factor ANOVA with a Tukey HSD; p-value = 0.0026933), while no difference was observed between the two regions on bare substrate (Fig. 6). Although a two-factor ANOVA only revealed differences in N content for growth region and not substrate type, the alga did demonstrate significant difference between the alga’s marginal samples on O. annularis when compared to the non-marginal replicates for both substrate types. Additionally, there was a nearly significant difference between the N content of R. textilis on O. annularis (mean % N ± SEM = 0.462 % ± 0.022) and bare substrate (mean % N ± SEM = 0.395 % ± 0.025) when isolating marginal replicates (one-factor ANOVA; p-value = 0.0671), while N percentages were nearly equivalent for the non-marginal thalli across substrate type.

Fig. 6: Boxplot of the % N from R. textilis growing on O. annularis (n = 10 colonies) and bare substrate (i.e., control; n = 10 rocks), with a marginal and non-marginal samples collected from each substrate type. A two-factor ANOVA found significant differences between growth region (p-value = 0.000521), but not between substrate types or the interaction effect of the two factors. Letters denote significant differences from a Tukey’s post hoc test.

The C:N ratio was significantly lower on O. annularis colonies (mean C:N ± SEM = 15.21 ± 0.47) when compared to bare substrate (mean C:N ± SEM = 17.72 ± 0.57), with no differences detected for growth region or the interaction of the main effects (Fig. 7). Since the N percentages of the alga’s non-marginal samples were consistent across substrate types, the disparity between the C:N was likely driven by higher carbon content observed in the alga growing on bare substrate (mean % C ± SEM = 5.954% ± 0.479) versus the coral colonies (mean % C ± SEM = 5.030% ± 0.331; Fig. 8). In contrast, the C content was fairly similar in the alga’s apical region for each substrate (control substrate = 6.861% ± 0.413; coral substrate = 6.9103% ± 0.320; Fig. 8); indicting that the difference in C:N may be attributed to the high N found in the alga encrusting over the O. annularis.

Fig. 7: Boxplot of the C:N from R. textilis growing on O. annularis (n = 10 colonies) and bare substrate (i.e., control; n = 10 rocks), with a marginal and non-marginal samples collected from each substrate type. A two-factor ANOVA found significant differences between substrate (p-value = 0.00223), but not between the growth regions or the interaction effect of the two factors. Letters denote significant differences from a two-factor ANOVA.

Fig. 8: Boxplot of the % C from R. textilis growing on O. annularis (n = 10 colonies) and bare substrate (i.e., control; n = 10 rocks), with a marginal and non-marginal samples collected from each substrate type. A two-factor ANOVA found significant differences between growth region (p-value = 0.00111), but not between substrate types or the interaction effect of the two factors. Letters denote significant differences from a Tukey’s post hoc test.

Brief Conclusion:

$\delta^{15}$N:

These results could support the hypothesis that Ramicrusta has the ability to remineralize coral nutrients as it colonizes coral colonies. Because of isotopic fractionation during metabolic processes in heterotrophic consumers, the significantly higher N signatures found in the alga overgrowing O. annularis could indicate the use of coral-derived nutrients. Futhermore, even under N saturated states, macroalgae generally assimiliate ¹⁴N and ¹⁵N in direct proportion (Fong et al., 2004; Fong et al., 2005), suggesting that the alga may be accessing alternative N sources based on substrate type.

It was anticipated that the highest N signatures would occur when the alga was directly interacting with live coral tissue. However, physiological stress disrupts the coral-symbiont relationship, as seen in bleaching events where corals expel symbionts under heat stress. Competition can also trigger the release of symbionts, disruption N recycling within the holobiont (Muscatine et al., 1978). Typically, corals exhibit low $\delta^{15}$N values (1-4%) because symbionts retain ¹⁴N (Muscatine et al., 1994). However, this expulsion releases the isotopically light N as ammonium into the water column (Muscatine et al. 1989). This ammonium leakage may explain the low $\delta^{15}$N values observed in Ramicrusta at the active site of coral overgrowth.

The apical regions of macroalgae thalli generally exhibit enriched $\delta^{15}$N values because the high N demand associated with new growth reduces N species selection, while the N supply available for the older portions of the thallus increases relative to it’s demand (Umezawa et al., 2007). However, we observed the opposite effects in Ramicrusta across both substrate types. Coral reef limestone function as a significant nitrate source for reef systems. Nitrate reduction to ammonium involves isotopic discrimination, potentially explaining the lightest $\delta^{15}$N values observed in marginal bare substrate samples due to preferential uptake of isotopically light compounds. Additionally, studies suggest macroalgae like Gracilaria vermiculophylla release dissolved organic nitrogen (DON) during senescence (Tyler et al., 2001). While less understood in macroalgae, diatoms like Thalassiosira pseudonana release ¹⁴N during incubation (Waser et al., 1998), possibly contributing to higher $\delta^{15}$N in the non-marginal (older, more established) portions of Ramicrusta from the bare substrate. Therefore, the gradient in $\delta^{15}$N values may reflect a combination of ¹⁴N selectivity in marginal alga samples and the ¹⁵N depleted release in non-marginal replicates collected from control substrates.

The $\delta^{15}$N thallus variation may be reduced as a result of coral-derived nutrients. Macroalgae preferentially assimilate ammonium over nitrate due to its lower energy cost, regardless of isotopic fractionation (Fong et al., 2004). However, the ammonium derived from corals may include both isotopically light and heavy components. Furthermore, the higher mean $\delta^{15}$N in alga samples at the coral interaction sites compared to alga along the marginal bare substrate suggests the incorporation of heavier N isotopes, which would in turn, reduce the $\delta^{15}$N variation in Ramicrusta thallus.

N content (To be continued..)

The striking disparity between the marginal and non-marginal N percentages in the alga overgrowing O. annularis colonies, compared to the control substrates, indicates a possible additional influx of nitrogen where the alga is actively encrusting over the coral.

Calcifying Macroalgae Isotopes:

The following compares the bulk stable isotope profiles of all calcifying macroalgae species. Results section will be drafted soon!

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